Calcium free subtilisin mutants

ABSTRACT

Novel calcium free subtilisin mutants are taught, in particular subtilisins which have been mutated to eliminate amino acids 75-83 and part or all of amino acids 1-22 (the N-terminal region) and which retain enzymatic activity and stability. Recombinant methods for producing the same and recombinant DNA encoding for such subtilisin mutants are also provided.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant No.GM42560 awarded by National Institute of Health.

GENERAL OBJECTS OF THE INVENTION

A general object of the invention is to provide subtilisin mutants whichhave been mutated such that they do not bind calcium.

Another object of the invention is to provide DNA sequences which uponexpression provide for subtilisin mutants which do not bind calcium.

Another object of the invention is to provide subtilisin mutants whichcomprise specific combinations of mutations which provide for enhancedthermal stability.

Another object of the invention is to provide a method for the synthesisof a subtilisin mutant which does not bind calcium-by the expression ofa subtilisin DNA which comprises one or more substitution', deletion oraddition mutations in a suitable recombinant host cell.

A more specific object of the invention is to provide class I subtilasemutants, in particular BPN′, mutants which have been mutated such thatthey do not bind calcium.

Another specific object of the invention is to provide DNA sequenceswhich upon expression result in class I subtilase mutants, and inparticular BPN′ mutants which do not bind calcium.

Another specific object of the invention is to provide a method formaking subtilisin I-S1 or I-S2 mutants, and in particular BPN′ mutantswhich do not bind calcium by expression of a class I subtilase mutantDNA sequence, and more specifically a BPN′ DNA coding sequence whichcomprises one or more substitution, addition or deletion mutations in asuitable recombinant host cell.

Yet another specific object of the invention is to provide mutantsubtilisin I-S1 or I-S2, and more specifically BPN′ mutants which do notbind calcium and which further comprise particular combinations ofmutations which provide for enhanced thermal stability, or which restorecooperativity to the folding reaction.

Yet a further object of the invention is to provide a mutant subtilisinprotein which has the calcium binding loop deleted (i.e. amino acids75-83) and deletion of amino acids in the N-terminal region (amino acids1-22). It was discovered that when the calcium binding loop was deleted,the N-terminal part of the molecule is no longer absolutely required forproper folding.

The subtilisin mutants of the present invention are to be utilized inapplications where subtilisins find current usage. Given that thesemutants do not bind calcium they should be particularly well suited foruse in industrial environments which comprise chelating agents, e.g.detergent compositions, which substantially reduces the activity ofwild-type calcium binding subtilisins.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to subtilisin proteins which have beenmodified to eliminate calcium binding. More particularly, the presentinvention relates to novel subtilisin I-S1 and I-S2 subtilisin mutants,specifically BPN′ mutants wherein the calcium A-binding loop has beendeleted, specifically wherein amino acids 75-83 have been deleted, andwhich may additionally comprise one or more other mutations, e.g.,subtilisin modifications, which provide for enhanced thermal stabilityand/or mutations which restore cooperativity to the folding reaction.Most particularly, the present invention relates to subtilisin proteinswhich have been modified to eliminate calcium binding and to delete theN-terminal region of the protein.

(2) Description of the Related Art

Subtilisin is an unusual example of a monomeric protein with asubstantial kinetic barrier to folding and unfolding. A well knownexample thereof, subtilisin BPN′ is a 275 amino acid serine proteasesecreted by Bacillus amyloliquefaciens. This enzyme is of considerableindustrial importance (such as for biodegradable cleaning agents inlaundry detergent) and has been the subject of numerous proteinengineering studies (Siezen et al., Protein Engineering 4:719-737(1991); Bryan, Pharmaceutical Biotechnology 3(B):147181 (1992); Wells etal., Trends Biochem. Sci. 13:291-297 (1988)). The amino acid sequencefor subtilisin BPN′ is known in the art and may be found in Vasantha etal., J. Bacteriol. 159:811-819 (1984). The amino acid sequence as foundtherein is hereby incorporated by reference [SEQUENCE ID NO:1].Throughout the application, when Applicants refer to the amino acidsequence of subtilisin BPN′ or its mutants, they are referring to theamino acid sequence as listed therein.

Subtilisin is a serine protease produced by Gram positive bacteria or byfungi. The amino acid sequences of numerous subtilisins are known.(Siezen et al., Protein Engineering 4:719-737 (1991)). These includefive subtilisins from Bacillus strains, for example, subtilisin BPN′,subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, andmesenticopeptidase. (Vasantha et al., “Gene for alkaline protease andneutral protease from Bacillus amyloliquefaciens contain a largeopen-reading frame between the regions coding for signal sequence andmature protein,” J. Bacteriol. 159:811-819 (1984); Jacobs et al.,“Cloning sequencing and expression of subtilisin Carlsberg from Bacilluslicheniformis, Nucleic Acids Res. 13:8913-8926 (1985); Nedkov et al.,”Determination of the complete amino acid sequence of subtilisin DY andits comparison with the primary structures of the subtilisin BPN′,Carlsberg and amylosacchariticus, Biol. Chem. Hoope-Seyler 366:421-430(1985); Kurihara et al., “Subtilisin amylosacchariticus,” J. Biol. Chem.247:5619-5631 (1972); and Svendsen et al., “Complete amino acid sequenceof alkaline mesentericopeptidase,” FEBS Lett. 196:228-232 (1986)).

The amino acid sequences of subtilisins from two fungal proteases areknown: proteinase K from Tritirachium albam (Jany et al., “Proteinase Kfrom Tritirachium albam Limber,” Biol. Chem. Hoppe-Seyler 366:485492(1985)) and thermomycolase from the thermophilic fungus, Malbrancheapulchella (Gaucher et al., “Endopeptidases: Thermomycolin,” MethodsEnzymol. 45:415433 (1976)).

These enzymes have been shown to be related to subtilisin BPN′, not onlythrough their primary sequences and enzymological properties, but alsoby comparison of x-ray crystallographic data. (McPhalen et al., “Crystaland molecular structure of the inhibitor eglin from leeches in complexwith subtilisin Carlsberg,” FEBS Lett., 188:55-58 (1985) and Pahler etal., “Three-dimensional structure of fungal proteinase K revealssimilarity to bacterial subtilisin,” EMBO J. 3:1311-1314 (1984)).

Subtilisin BPN′ is an example of a particular subtilisin gene secretedby Bacillus amyloliquefaciens. This gene has been cloned, sequenced andexpressed at high levels from its natural promoter sequences in Bacillussubtilis. The subtilisin BPN′ structure has been highly refined (R=0.14)to 1.3 Å resolution and has revealed structural details for two ionbinding sites (Finzel et al., J. Cell. Biochem. Suppl. 10A:272 (1986);Pantoliano et al., Biochemistry 27:8311-8317 (1988); McPhalen et al.,Biochemistry 27: 6582-6598 (1988)). One of these (site A) binds Ca²⁺with high affinity and is located near the N-terminus, while the other(site B) binds calcium and other cations much more weakly and is locatedabout 32 aa away (FIG. 1). In subtilisin BPN′, calcium binds to site Awith an affinity of −10⁷ M⁻¹ (Bryan et al, Biochemistry 31:4937-4945(1992)). By binding at a specific site in the tertiary structure,calcium contributes its binding energy to the stability of the nativestate and makes a large contribution to the overall free energy offolding (Schellman, Biopolymers 14:999-1018 (1975)). Structural evidencefor two calcium binding sites was also reported by Bode et al., Eur. J.Biochem. 166:673-692 (1987) for the homologous enzyme, subtilisinCarlsberg.

Further in this regard, the primary calcium binding site in all of thesubtilisins in groups I-S1 and I-S2 (Siezen et al., 1991, Table 7) areformed from almost identical nine residue loops in the identicalposition of helix C. X-ray structures of the I-S1 subtilisins BPN′ andCarlsberg, as well as the I-S2 subtilisin Savinase, have been determinedto high resolution. A comparison of these structures demonstrates thatall three have almost identical calcium A-sites.

The x-ray structure of the class I subtilase, thermitase fromThermoactinomyces vulgaris, is also known. Though the overall homologyof BPN′ to thermitase is much lower than the homology of BPN′ to I-S1and I-S2 subtilisins, thermitase has been shown to have an analogouscalcium A-site. In the case of thermitase, the loop is a tenresidue-interruption at the identical site in helix C.

Calcium binding sites are common features of extracellular microbialproteases probably because of their large contribution to boththermodynamic and kinetic stability (Matthews et al., J. Biol. Chem.249:8030-8044 (1974); Voordouw et al., Biochemistry 15:3716-3724 (1976);Betzel et al., Protein Engineering 3:161-172 (1990); Gros et al., J.Biol. Chem. 266:2953-2961 (1991)). The thermodynamic and kineticstability of subtilisin is believed to be necessitated by the rigors ofthe extracellular environment into which subtilisin is secreted, whichby virtue of its own presence is protease-filled. Accordingly, highactivation barriers to unfolding may be essential to lock the nativeconformation and prevent transient unfolding and proteolysis.

Unfortunately, the major industrial uses of subtilisins are inenvironments containing high concentrations of metal chelators, whichstrip calcium from subtilisin and compromise its stability. Therate-determining step in the inactivation of subtilisin, under stronglychelating conditions, is the loss of calcium from site A (Bryan et al,Biochemistry 31:4937-4945 (1992); Voordouw, Biochem. 15:3716-3724(1976)). Presumably, natural protein structures evolve to be stable inthe native environment, therefore alternative structural solutionsshould be possible which are better suited to new environments. Itwould, therefore, be of great practical significance to create a highlystable subtilisin which is independent of calcium.

The present inventors have previously used several strategies toincrease the stability of subtilisin to thermal denaturation by assumingsimple thermodynamic models to approximate the unfolding transition(Pantoliano et al., Biochemistry 26:2077-2082 (1987); Pantoliano et al.,Biochemistry 27:8311-8317 (1988); Pantoliano et al., Biochemistry28:7205-7213 (1989); Rollence et al., CRC Crit. Rev. Biotechnol.8:217-224 (1988). However, improved subtilisin mutants which are stablein industrial environments, e.g., which comprise metal chelators, andwhich do not bind calcium, are currently not available.

OBJECTS AND SUNIMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide mutated ormodified subtilisin enzymes, e.g., class I subtilases, which have beenmodified to eliminate calcium binding. As used in this invention, theterm “mutated or modified subtilisin” is meant to include any serineprotease enzyme which has been modified to eliminate calcium binding.This includes, in particular, subtilisin BPN′ and serine proteases whichare homologous to subtilisin BPN′, in particular class I subtilases.However, as used herein, and under the definition of mutated or modifiedsubtilisin enzyme, the mutations of this invention may be introducedinto any serine protease which has at least 50%, and preferably 80%amino acid sequence identity with the sequences-referenced above forsubtilisin BPN′, subtilisin Carlsberg, subtilisin DY, subtilisinamylosacchariticus, mesenticopeptidase, thermitase, or Savinase and,therefore, may be considered homologous.

The mutated subtilisin enzymes of this invention are more stable in thepresence of metal chelators and may also comprise enhanced thermalstability in comparison to native or wild-type subtilisin. Thermalstability is a good indicator of the overall robustness of a protein.Proteins of high thermal stability often are stable in the presence ofchaotropic agents, detergents, and under other conditions, whichnormally tend to inactivate proteins. Thermally stable proteins are,therefore, expected to be useful for many industrial and therapeuticapplications in which resistance to high temperature, harsh solventconditions or extended shelf-life is required.

It has been further discovered that combining individual stabilizingmutations in subtilisin frequently results in approximately additiveincreases in the free energy of stabilization. Thermodynamic stabilityhas also been shown to be related to resistance to irreversibleinactivation at high temperature and high pH. The single-site changes ofthis invention individually do not exceed a 1.5 Kcal/mol contribution tothe free energy of folding. However, these small incremental increasesin the free energy of stabilization result in dramatic increases inoverall stability when mutations are combined, since the total freeenergy of folding for most proteins is in the range of 5-15 Kcals/mol(Creighton, Proteins: Structure and Molecular Properties, W.H. Freemanand Company, New York (1984)).

X-ray crystallographic analysis of several combination mutants revealsthat conformational changes associated with each mutation tend to behighly localized with minimal distortion of the backbone structure.Thus, very large increases in stability can be achieved with no radicalchanges in the tertiary protein structure and only minor independentalterations in the amino acid sequence. As previously suggested (Holmeset al, J. Mol. Biol. 160:623 (1982)), contributions to the free energyof stabilization can be gained in different ways, including improvedhydrogen bonding and hydrophobic interactions in the folded form anddecreased chain entropy of the unfolded enzyme. This is significantsince thermostable enzymes generally have more extended half-lives atbroader temperature ranaes, thereby improving bio-reactor and shelf-lifeperformance.

As noted supra, the invention provides subtilisin mutants which compriseone or more deletion, substitution or addition mutations which providefor the elimination of calcium binding. Preferably, this will beeffected by deletion, substitution or insertion of amino acids into thecalcium A-site, which in the case of class I subtilases comprises 9amino acid residues in helix C. In the case of subtilisin BPN′, thesubtilisin mutants will preferably comprise one or more addition,deletion or substitution mutations of the amino acids at positions75-83, and most preferably will comprise the deletion of amino acids75-83, of SEQUENCE ID NO: 1. The deletion of amino acids 75-83 has beendiscovered to effectively eliminate calcium binding to the resultantsubtilisin mutant while still providing for subtilisin BPN′ proteinshaving enzymatic activity.

Such subtilisin mutants lacking amino acids 75-83 of SEQUENCE ID NO: 1may further include one or more additional amino acid mutations in thesequence, e.g., mutations which provide for reduced proteolysis. It isanother object of the invention to produce subtilisin mutants lackingcalcium binding activity which have been further mutated to restorecooperativity to the folding reaction and thereby enhance proteolyticstability. It is another object of the invention to provide thermostablesubtilisin mutants which further do not bind calcium and comprisespecific combinations of mutations which provide for substantiallyenhanced thermal stability.

In particular, the subtilisin mutants of the present invention willinclude subtilisins from Bacillus strains, such as subtilisin BPN′,subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus andsubtilisin mesenticopeptidase, which comprise one or more deletion,substitution or addition mutations.

The present invention further provides for subtilisin mutants lackingamino acids 75-83 of SEQUENCE ID NO: 1, which have new protein-proteininteractions engineered in the regions around the deletion leading tolarge improvements in stability. More specifically, mutations at tenspecific sites in subtilisin BPN′ and its homologues are provided, sevenof which individually, and in combination, have been found to increasethe stability of the subtilisin protein. Improved calcium-freesubtilisins are thus provided by the present invention.

The present invention further provides for subtilisin mutants lackingamino acids 75-83 of Sequence ID NO:1, which also have amino acidsdeleted in the N-terminal region (amino acids 1-22). More specifically,the present invention provides for deletions of part of all of thisN-terminal region-together with further stabilizing mutations, asdiscussed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. X-ray Crystal structure of S15 subtilisin

A. ct-carbon plot shows the positions of mutations as noted. Thenumbering of wild type subtilisin is kept. Dotted spheres show theposition of calcium at the weak ion binding site (B-site) and the formerposition of the high affinity, binding site (A-site). The A-site loop(dashed line) is absent in this mutant. N- and C-termini are indicated.The N-terminus is disordered (dotted line).

B. Close-up view of the A-site deletion., The loop from S12 subtilisinis shown as a dotted line with the continuous helix of S15. Superimposedis the 3* sigma difference electron density (FO12-FO15., phases fromS15) showing the deleted A-site loop.

FIG. 2. X-ray crystal structure of the calcium A-site region of S12subtilisin. Calcium is shown as a dotted sphere with one-half the vander Waals radius. Dashed lines are coordination bonds, while dottedlines represent hydrogen bonds under 3.2 Å.

FIG. 3. Differential Scanning Caloriinetry. The calorimetric scans ofapo-S12 (T_(m)=63.5° C.) and S15 (T_(m)=63.0° C.) are shown.Measurements were performed with a Hart 7707 DSC (differential scanningcalorimetry) heat conduction scanning microcalorimeter as described(Pantoliano et al., Biochemistrv 28:7205-7213 (1989)). Sample conditionswere 50 mM of glycine, a pH of 9.63, a scan rate of 0.5° C./min. Excessheat capacity is measured in units of μJ/°. The calorimeter ampoulescontained 1.78 mg of protein.

FIG. 4. Titration calorimetry of subtilisin S11. The heat of calciumbinding for successive additions of calcium are plotted vs. the ratio of[Ca]/[P]. The data are best fit by a calculated binding curve assuming abinding constant of 7×10⁶ and ΔH equal to 11.3 kcal/mol using equation(1) from the text. For comparison, calculated curves assumingK_(a)=1×10⁶ and 1×10⁸ are also shown. In this titration, [PI=100 μM andthe temperature was 25° C.

FIG. 5. Kinetics of calcium dissociation from subtilisin S11 as afunction of temperature. 1 μM subtilisin S11 was added to 10 μM Quin2 attime=0. Calcium dissociates from subtilisin and binds to Quin2 until anew equilibrium is achieved. The rate of calcium dissociation isfollowed by the increase in fluorescence of Quin2 when it binds tocalcium.

A. The log of the percent of the protein bound to calcium is plotted vs.time. The kinetics of dissociation at four temperatures are shown. Thedissociation follows first order kinetics for the first 25% of thereaction. As this is well before equilibrium is approached,reassociation of calcium can be neglected.

B. Temperature dependence of the rate of calcium dissociation from S15subtilisin in the presence of excess Quin2, pH 7.4 and over atemperature range of 25-45° C. The natural log of the equilibriumconstant for the transition state (calculated from the Eyring equation)is plotted vs. the reciprocal of the absolute temperature. The line isfit according to equation (3) in the text with T₀=298 K.

FIG. 6. Analysis of subtilisin refolding monitored by circular dichroism(CD).

A. CD spectra are shown for S15 as follows: (1) S15 in 25 mM H₃PO₄ at pH1.85; (2) S15 denatured at pH 1.85 and then neutralized to pH 7.5 by theaddition of NAOH; (3) S15 denatured at pH 1.85 and neutralized to pH7.5, 30 minutes after the addition of KCI to 0.6 M; and (4) Native S15subtilisin. Protein concentrations of all samples was 1 μM.

B. Kinetics of refolding of S15. Samples were denatured at pH 1.85 andthen the pH was adjusted to 7.5. At time 0, KCI was added to thedenatured protein. Recovery of native structure was followed at 222 runat KCI concentrations of 0.3 M and 0.6 M. The 0.6 M sample after 30minutes of refolding was then used to record the corresponding spectrumin part A.

FIG. 7. Kinetics of refolding of S15 as a function of ionic strength.

A. The log of the percent unfolded protein is plotted vs. time. Thekinetics of refolding are shown at four ionic strengths. The amount ofrefolding was determined by circular dichroism (CD) from: the increasein negative ellipticity at 222 nm. 100% folding is determined from thesignal at 222 nm for native S15 at the same concentration and 0% foldingis determined from the signal for acid-denatured S15. The refoldingapproximately follows first order kinetics for the first 90% of thereaction. Refolding was carried out at 25° C.

B. The log of first order rate constants for refolding obtained by CD orfluorescence measurements at 25° C. were plotted as a function of log ofionic strength. Ionic strength was varied from I=0.25 to I=1.5. The rateof refolding increases linearly with log I. A ten-fold increase in Iresults in an approximately 90-fold increase in the refolding rate.

FIG. 8. Temperature dependence of the refolding rate of S15 subtilisinin 0.6 M KCI, 23 nM KP04 pH 7.3. The natural log of the equilibriumconstant for the transition state (calculated from the Eyring equation)is plotted vs. the reciprocal of the absolute temperature. The line isfit according to equation 3 in the text with T₀=298 K.

FIG. 9. X-ray crystal structure of the weak ion binding region of S15subtilisin. Coordination bonds are shown as dashed lines. Note thepreponderance of charged amino acids.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As discussed supra, calcium binding contributes substantially to thethermodynamic and kinetic-stability of extracellular microbialproteases. Moreover, with respect to subtilisin, hiah activationbarriers to unfolding may be essential to retain the native conformationand to prevent transient unfolding and proteolysis given theprotease-filled environment where subtilisin is secreted and as a resultof auto-degradation. The unfolding reaction of subtilisin can be dividedinto two parts as follows:${N\left( {Ca}_{2} \right)}\overset{\Delta \quad g_{1}}{}{N({Ca})}\overset{\Delta \quad g_{2}}{}N\overset{\Delta \quad g_{3}}{}U$

where N(Ca₂) is the native form of subtilisin with calcium bound to bothsites; N(Ca) is the native form of subtilisin with calcium bound to thehigh affinity calcium-binding site A (Finzel et al., J. Cell. Biochem.Suppl. 10A:272 (1986); Pantoliano et al., Biochemistry 27:8311-8317(1988); McPhalen et al., Biochemistry 27:6582-6598 (1988)); N is thefolded protein without calcium bound; and U is the unfolded protein. Thetotal free energy of unfolding is therefore equal to Δg₁+Δg₂+Δg₃. Fromthe binding constant, one can calculate the contribution of calcium tothe free energy of subtilisin folding from the following equation:

ΔG _(binding)=−RT ln(1+K_(a)[Ca]).

Thus, the contribution of site A to the stability of subtilisin in 10 mMcalcium is 6.6 kcal/mol at 25° C. The contribution of calcium binding tosite B in 10 mM calcium and 50 mM sodium is only 0.2 kcal/mol. Thisanalysis is at odds with earlier studies which concluded that calciumbinding to site B is responsible for the large decrease in theinactivation rate of subtilisin in the presence of millimolarconcentrations of calcium (Braxton & Wells, Biochem. 31:7796-7801(1992); Pantoliano et al, Biochem. 27: 8311-8317 (1988)).

Subtilisin is a relatively stable protein whose stability is in largepart mediated by the high affinity calcium site (Voordouw et al.,Biochemistry 15:3716-3724 (1976); Pantoliano et al., Biochemistry27:8311-8317 (1988)). The melting temperature of subtilisin at pH 8.0 inthe presence of μmolar concentrations of calcium is approximately 75° C.and approximately 56° C. in the presence of excess EDTA (Takehashi etal., Biochemistry 20:6185-6190 (1981); Bryan et al., Proc. Natl. Acad.Sci. USA, 83:3743-3745 (1986b)). Previous calorimetric studies of thecalcium-free (apoenzyme, i.e., protein portion of enzyme) form ofsubtilisin indicated that it is of marginal stability at 25° C. with aAG of unfolding of <5 kcal/mol (Pantoliano et al., Biochemistry28:7205-7213 (1989)). Because calcium is such an integral part of thesubtilisin structure, the apoenzyme is thought to be a foldingintermediate of subtilisin.

In order to independently examine the two phases of the folding process,the present inventors constructed a series of mutant subtilisins. First,all proteolytic activity was eliminated in order to preventauto-degradation from occurring during the unfolding and refoldingreactions. This may be accomplished, for example, by converting theactive-site serine 221 to cysteine.¹ This mutation has little effect onthe thermal denaturation temperature of subtilisin, but reducespeptidase activity of subtilisin by a factor of approximately 3×104(Abrahmsen et al., Biochemistry 30:4151-4159 (1991)). This mutant,therefore, allows the folding of subtilisin to be studied without thecomplications of proteolysis. In the present specification, a shorthandfor denoting amino acid substitutions employs the single letter aminoacid code of the amino acid to be substituted, followed by the numberdesignating where in the amino acid sequence the substitution will bemade, and followed by the single letter code of the amino acid to beinserted therein. For example, S221C denotes the substitution of serine221 to cysteine. The subtilisin mutant with this single amino acidsubstitution is denoted subtilisin S221C. The resulting S221C subtilisinmutant is designated S1.

¹ The S221A mutant was originally constructed for this purpose. Themature form of this mutant was heterogeneous on its N-terminus, however,presumably due to some incorrect processing of the pro-enzyme.

The subtilisin may be further mutated in order to make the relativelyunstable apoenzyme easier to produce and purify. Versions of S1 withthree or four additional mutations, for example, M50F, Q206I, Y217K andN218S, may also be employed in the method of the present invention. Suchfurther mutations cumulatively increase the free energy of unfolding by3.0 kcal/mol and increase the thermal denaturation temperature of theapoenzyme by 11.5° C. (Pantoliano et al., Biochemistry 28:7205-7213(1989)). The mutant containing the M50F, Q206I, Y217K, N218S and S221Cmutations is designated S11 and the mutant containing the M50F, Y217K,N218S and S221C is designated S12.²

² The specific activities of S11, S12 and S15 against the syntheticsubstrate, SAAPFna, are the same. (S.A.=0.0024 U/mg at 25° C., pH 8.0).These measurements were performed on protein freshly purified on amercury affinity column.

In order to produce a subtilisin BPN′ protein lacking calcium bindingactivity, the present inventors elected to delete the binding loop inthe calcium Asite to engineer a novel calcium-free subtilisin protein.This loop comprises an interruption in the subtilisin BPN′ α-helixinvolving amino acids 63-85 of SEQUENCE ID NO:1 (McPhalen and James1988). Residues 75-83 of the subtilisin BPN′ protein form a loop whichinterrupts the last turn of the 14-residue alpha helix involving aminoacids 63-85 [SEQUENCE ID NO: 1].³ Four of the carbonyl oxygen licands tothe calcium are provided by a loop composed of amino acids 75-83[SEQUENCE ID NO: 1]. The geometry of the ligands is that of a pentagonalbipyramid whose axis runs through the carbonyls of amino acids 75 and79. On one side of the loop is the bidentate carboxylate (D41), while onthe other side is the N-terminus of the protein and the side chain ofQ2. The seven coordination distances ran-e from 2.3 to 2.6 A, theshortest being to the aspartyl carboxylate. Three hydro-en bonds linkthe N-terminal seament to loop residues 78-82 in parallel-betaarrangement. A high affinity calcium binding site is a common feature ofsubtilisins-which make larce contributions to their high stability. Bybinding at specific sites in the subtilisin tertiary structure, tertiarystructure, calcium contributes its binding free energy to the stabilityof the native state. In the present invention, site-directed mutagenesiswas used to delete amino acids 75-83 of SEQUENCE ID NO: 1, which createsan uninterrupted helix and abolishes the calcium binding potential atsite A (FIG. 1A and 1B).

³ This set of nine residues was chosen for deletion, as opposed to 74-82(those actually belonging to the loop) out of preference for Ala 74rather than Gly 83 in the resulting continuous helix. Alanine has ahigher statistical likelihood for occurrence in α-helix, due toglycine's broader range of accessible backbone conformations.

The present inventors believed that a stabilization strategy basedaround calcium binding would allow survival in the extracellularenvironment. Since the major industrial uses of subtilisins are inenvironments containing high concentrations of metal chelators, it wasof areat practical significance for the present inventors to produce astable subtilisin which is independent of calcium and, therefore,unaffected by the presence of metal chelating agents. Thus, stabilizingmutations in subtilisin can be classified into three groups: 1)stabilizing only in calcium, 2) stabilizing only in chelants; 3)stabilizing in both conditions (Table 1). From this partitioning it isevident that the mechanism of thermal inactivation differs depending onwhether the calcium sites are occupied. To understand why this is so,one must understand how the kinetics of inactivation are related to thekinetics of unfolding and how the kinetics of unfolding are related tothe kinetics of calcium loss.

While the present inventors chose to eliminate calcium binding by theremoval of these amino acids (i.e. amino acids at positions 75-83), itshould be possible to eliminate calcium binding by other mutations,e.g., substitution of one or more of the amino acids at positions 75-83with alternative amino acids and by insertion, substitution and/ordeletion of amino acids proximate to positions 75-83. This may also beaccomplished by site-specific mutagenesis.

Additionally, because this loop is a common feature of subtilisins, itis expected that equivalent mutations for other subtilisins, inparticular class I subtilases, e.g., by site-specific mutagenesis, willlikewise eliminate calcium binding and provide for enzymatically activemutants.

In particular, the present inventors synthesized by site-specificmutagenesis three subtilisin BPN′ DNA's which have been mutated toeliminate amino acids 75-83 involved in calcium binding and whichfurther comprise additional substitution mutations. These mutatedsubtilisin BPN′ DNA's, upon expression of the DNA, provide forsubtilisin proteins having enhanced thermal stability and/or which areresistant to proteolysis.

The specific subtilisin BPN′ mutants synthesized by the presentinventors are designated in this application as S15, S39, S46, S47, S68,S73, S79, S86, S88 and pS149. The specific point mutations set forth inthe present application identify the particular amino acids in thesubtilisin BPN′ amino acid sequence, as set forth in SEQUENCE ID NO: 1,that are mutated in accordance with the present invention. For example,the S15 mutant comprises a deletion of amino acids 7583 and additionallycomprises the following substitution mutations: S221C, N218S, M50F andY217K. The S39 mutant similarly comprises a deletion of amino acids75-83 and additionally comprises the following substitution mutations:S221C, PSA, N218S, M50F and Y217K. The S46 mutant comprises a deletionof amino acids 75-83 and additionally, comprises the followingsubstitution mutations: M50F, Y217K and N218S. The S47 mutant similarlycomprises a deletion of amino acids 75-83 and additionally comprises thefollowing substitution mutations: P5A, N218S, M50F and Y217K. The S68mutant comprises a deletion of amino acids 75-83 and additionallycomprises the following substitution mutations: P5S, N218S, M50F andY217K. The S73 mutant comprises a deletion of amino acids 75-83 as wellas the following substitution mutations: Q2K, M50F, A73L, Q206V, Y217Kand N218S. The S79 mutant comprises a deletion of amino acids 75-83 andadditional comprises the following substitution mutations: Q2K, M50F,A73L, Q206C, Y217K and N218S,. The S86 mutant comprises a deletion ofamino acids 75-83 as well as the following substitution mutations: Q2K,S3C, M50F, A73L, Q206C, Y217K and N218S. The S88 mutant comprises adeletion of amino acids 75-83 as well as the following substitutionmutations: Q2K, S3C, P5S, K43N, M50F, A73L, Q206C, Y217K, N218S, andQ271E. Finally, the pS149 mutant comprises a deletion of amino acids75-83 as well as the mutations present in the S88 mutant and thefollowing substitution mutations: S9A, I31L, E156S, G166S, G169A, S188P,N212G, K217L and T254A. The specific activities of the proteolyticallyactive S46, S47, S68, S73, S79, S86, S88 and pS149 subtilisins have beenfound to be similar or enhanced in relation to the wild-type enzyme.

The inventors also consider as part of their invention Δ75-83 subtilisinmutants which contain one or more of the following mutations: Q2K, S3C,P5S, K43N, M50F, A73L, Q206C, Y217K, N218S, Q271E, S9A, I31L, E156S,G166S, G169A, S188P, N212G, K217L and T254A. Furthermore, applicantsconsider as part of their invention Δ75-83 subtilisin mutants whichcontain one or more substitutions selected from the group consisting ofS9A, I31L, E156S, G166S, G169A, S188P, N212G, K217L, L1261, M222Q andT254A, and optionally together with at least one or more substitutionsselected from the group consisting of Q2K, S3C, P5S, K43N, M50F, A73L,Q206C, Y217K, N218S, and Q271E.

The inventors further consider to be part of their invention Δ75-83subtilisin mutants which have a part or all of the N-terminal regiondeleted (aa 1-22). An example of such a mutant subtilisin protein isS176, which has the Δ75-83 deletion and a deletion of amino acids 1-22.Another example of such a mutant subtilisin protein is S177, which hasthe Δ75-83 deletion as well as a deletion of amino acids 1-17. One ofskill in the art could easily determine other deletions within theN-terminal region which could be made to the subtilisin mutant proteinsof the present invention. Such mutations should preserve the structuralintegrity and stability of the subtilisin protein. One of skill in theart could determine which mutations within the N-terminal region arepreferable by measuring the stability and structural integrity of themutant using technologies such as circular dichroism.

These mutant subtilisins (with the Δ75-83 deletion and the deletion ofpart or all of the N-terminal region) may also have any of thestabilizing mutations discussed above (i.e. S221C, Y217K, P5A, M50F,N218S, P5S, Q2K, A73L, Q206V, Q206C, S3C, K43N, Q271E, S9A, I31L, E156S,G166S, G169A, S188P, N212G, K217L, T254A, L1261 and M222Q). Mostpreferably, the stabilizing mutations are present in the following aminoacid positions: 41-45; 70-74; 86-87; 181-184; 200-214; 226-237; and267-275.

The various Δ75-83 subtilisins which were synthesized by the inventorsare shown in Tables 1 and 2, below. The particular points of mutation inthe amino acid sequence of subtilisin BPN′ amino acid sequence, as setforth in SEQUENCE ID NO:1, are identified. The synthesis of thesemutants is described in more detail infra.

TABLE I Subtilisin Mutations S221C P5A Δ75-83 N218S M50F Q2061 Y217KQ271E Q2K A73L K43N Q206C S3C BPN′ − − − − − − − − − − − − − S1* + − − −− − − − − − − − − S11* + − − + + + + − − − − − − S12* + − − + + − + − −− − − − S15* + − + + + − + − − − − − − S39 + + + + + − + − − − − − − S46− − + + + − + − − − − − − S47 − + + + + − + − − − − − − S68 − P5S + + +− + − − − − − − S73 − − + + + − + − − − − − − S79 − − + + + − + + + +− + − S86 − − + + + − + + + + − + + S88 − P5S + + + − + + + + + + + Theplus signs show that a subtilisin contains a particular mutation. X-raycrystal structure of wild type, S12 and S15 have been determined to1.8Å. *S1, S11, S12, S15 and S39 are low activity mutants constructed toaid in the evaluaton of structure and conformational stability. S88 S9A131L E156S G166S G169A N212G S188P K217L T254A pS149*88 + + + + + + + + + *pS149 contains all of the mutations present inS88, in addition to the mutations depicted in the table.

In order to understand the contribution of calcium binding to thestability of subtilisin, the thermodynamics and kinetics of calciumbinding to the high affinity calcium A-site were measured bymicrocalorimetry and fluorescence spectroscopy. Calcium binding is anenthalpically driven process with an association constant (K_(a)) equalto 7×10⁶ M⁻¹. The kinetic barrier to calcium removal from the A-site (23kcal/mol) is substantially larger than the standard free energy ofbinding (9.3 kcal/mol). The kinetics of calcium dissociation fromsubtilisin (e.g, in excess EDTA) are accordingly very slow. For example,the half-life (t_(1/2)) of calcium dissociation from subtilisin, i.e.,the time for half of the calcium to dissociate from subtilisin, is 1.3hours at 25° C.

X-ray crystallography shows that except for the region of the deletedcalcium-binding loop, the structure of the subtilisin mutants and thewild type protein are very similar. The N-terminus of the wild-typeprotein lies beside the site A loop and furnishes one calciumcoordination ligand, the side chain oxygen of Q2. In Δ5-83 subtilisin,the loop is -one, leaving residues 1-4 disordered. These first fourresidues are disordered in the X-ray structure since all itsinteractions were with the calcium loop. N-terminal sequencing confirmsthe first four amino acids are pesent, confirming that processing occursat the normal site. The helix is shown to be uninterrupted and showsnormal helical geometry over its entire length. X-ray crystallographyfurther shows that the structures of subtilisin with and without thedeletion superimpose with a root mean square (r. m. s.) differencebetween 261 α-carbon positions of 0.17 Å and are remarkably similarconsidering the size of the deletion. Diffuse difference density andhigher temperature factors, however, indicate some disorder in the newlyexposed residues adjacent to the deletion.

While the elimination of calcium binding is advantageous since itproduces proteins that are more stable in the presence of metalchelators, it is disadvantageous in at least one respect. Specifically,the elimination of the calcium loop without any other compensatingmutations results in the destabilization of the native state relative tothe partially folded states and, therefore, a loss of cooperativity infolding. The present inventors thus sought to further geneticallyengineer the subtilisin S15 BPN′ protein lacking amino acids 75-83 inorder to restore cooperativity to the folding reaction. In most welldesigned proteins all parts of the molecule are interdependent, makingthe unfolding reaction highly cooperative. Cooperativity of the foldingreaction allows proteins to achieve sufficient stabilities of the nativestate for proper function since the overall stability of the nativeconformation is roughly the sum of all local interactions.

Therefore, while the Δ75-83 subtilisin is an example of an engineeredsubtilisin which is active and stable in the absence of calcium, thepresent inventors sought to improve this protein by further mutation.The design of a particular highly stable calciwn-free subtilisin relieson an iterative engineering cycle. The present inventors found that therequisite first step in the cycle was to greatly diminish theproteolytic activity of subtilisin. This is necessary because calciumcontributes greatly to the conformational stability of subtilisin andthe early versions of calcium-free subtilisin are susceptible toproteolysis. After reducing the susceptibility to proteolysis, the nextstep in the cycle was to eliminate sequences essential for calciumbinding, i.e., the A-site. Although the S15 Δ75-83 subtilisin is muchless stable than the wild type subtilisin in the presence of calcium,this mutant is more stable than wild type subtilisin in the presence ofthe metal chelator EDTA.

Accordingly, the third step was to improve the stability of thecalciumfree subtilisin protein. To improve the stability of calcium-freesubtilisin, the present inventors next tried to create a home for thedisordered N-tenninal residues. In order to create a highly stablecalcium-free subtilisin, the N-terminal part of the protein which isdestabilized by the deletion of the calcium A-loop may be modified. Forexample, the N-terminus which is disordered may be deleted or extended.This, however, is problematic because the requirements for processingthe propeptide from the mature protein are not known. It is known,however, that the processing site is not determined by amino acidsequence since mutants Y1A (the C-terminus of the propeptide), A1C andQ2R do not alter the site of cleavage. It is also known that the nativestructure of the N-terminus in subtilisin does not determine thecleavage site because the A75-83 variants are processed correctly. Sinceit is not yet known how to alter the processing site, interactions withthe existing N-terminus may be optimized.

Examination of the structure of S15 subtilisin revealed numerouspossibilities for improving stability of the mutant enzyme. The regionsof the structure most affected by the deletion are the N-terminal aminoacids 1-8, the 36-45 ω-loop, the 70-74 α-helix the 84-89 helix turn andthe 202-219 β-ribbon. As previously stated, the first four residues inΔ75-83 subtilisin are disordered in the x-ray structure since all itsinteractions had been with the calcium loop. N-terminal sequencingshows, however, that the first four amino acids are present confirmingthat processing occurs at the normal site. Other than the N-terminus,there are three other residues whose side chain conformations aredistinctly different from wild type. Y6 swings out of a surface nicheinto a more solvent-exposed position, as an indirect effect of thedestabilization of the N-terminus. D41, a former calcium ligand, andY214 undergo a coordinated rearrangement, forming a new hydrogen bond.The B-factors of all three residues increase significantly due to thedeletion of amino acids 75-83. In addition, S87 and A88 do not changeconformation but exhibit significantly increased B-factors. P86terminates the a-hela from which the calcium loop was deleted. In viewof the above, other mutations at one or more of the above mentionedsites, or at the amino acids proximate thereto, will provide forsubtilisin BPN′ mutants comprising greater enzymatic activity orincreased stability.

There are several logical strategies for remodeling this region of theprotein to produce subtilisin BPN′ mutants comprising greater enzymaticactivity or increased stability. Since the N-terininal four amino acidsare disordered in the x-ray structure, one possible approach would be todelete them from the protein. The requirements for processing thepropeptide from the mature protein are not understood, however.Inserting or deleting amino acids from the N-terminal region is,therefore, problematic. For this reason insertions and deletions in theN-terminal region were avoided in favor of amino acid substitutions.Many of the original amino acids in the above described regions ofsubtilisin which interacted with the amino acids 75-83 loop can beassumed to no longer be optimal. It was, therefore, possible to increasethe stability of the molecule by substituting, deleting or adding atleast one amino acid at positions whose environment was changed by the75-83 deletion.

The first attempt was to mutate the proline at position 5 to alanine tocreate more flexibility at position 5. This increased flexibility allowsthe N-terminus to try to find a unique position along the new surface ofthe protein, created by deletion of the calcium loop. Once theN-terminus assumes a unique location its local interactions may then beoptimized.

The P5A mutation was made to try to create more flexibility for theN-terminus and allow it to find a unique position along the new surfaceof the protein that was created by deletion of the calcium loop. In thenative structure, the first five amino acids are in an extendedconformation and form β-pair hydrogen bonds with the calcium loop aswell as the Q2 side chain interaction with the calcium. The proline atposition 5, which is conserved among seven bacterial subtilisins whichhave a homologous calcium A-site, may help stabilize the extendedconformation. The P5A mutation in Δ75-83 subtilisin should thus resultin an increase in the cooperativity of the unfolding reaction. The X-raystructure′of this variant has been determined to 1.8 Å.

In toto, the present inventors selected amino acids at ten differentpositions whose environment had changed substantially for substitution.A mutagenesis and screening procedure was developed in order to screenall possible substitutions at a particular site. The technique forgenerating and screening subtilisin variants involves in vitromutagenesis of the cloned subtilisin gene, expression of the mutatedgenes in B. subtilis, and screening for enhanced stability.

For example, site-directed mutagenesis was performed on the S46subtilisin gene using oligonucleotides which were degenerate at onecodon. The degenerate codon contained all combinations of the sequenceNNB, where N is any of the four nucleotides and B is T, C or G. The 48codons represented in this population encode for all twenty amino acidsbut exclude the ochre and umber termination codons. The mutagenizedgenes were used to transform B. subtilis. Examples of particularmutations are shown in Table II as follows:

TABLE II Site-directed mutagenesis Stabilizing Region of protein Sitemutations Mutagenic Oligonucleotide N-terminus Q2 K, W, L AC GCG TAC GCGNNB TCC GTG CCT TAC S3 C* GCG TAC GCG AAG MMB GTG CCT TAC CG V4 none CGCG AAG TCC NNB CCT TAC GGC G P5 S CAG TCC GTG NNB TAC GGC GTA TC 36-44omega loop D41 A GAT TCT TCT CAT CCT NNB TTA AAG GTA GC K43 R, N CAT CCTGAT TTA NNB GTA GCA GGC GG 63-85 α-helix A73 L, Q GGC AVA GTT NNB GCTGTT GCG A74 none C ACA GTT GCG NNB GTT GCG CCA AG 202-220 β-ribbon Q206I, V, W, C* C GTA TCT ATC MMB AGC ACG CTT CC Y214 none CCT GGA AAC AAANTN GGG GCG AAA TC *Double cysteine mutations at positions 3 and 206have been found to be as stabilizing as a disulfide bond.

To have a 98% chance of finding tryptophan, glutamine, glutamate ormethionine in the mutant population, one must screen about 200 mutantclones. Each of those codons is represented by only one of the 48 codonscontained in the population of sequences NNB. Codons for all other aminoacids are represented by at least two codons in the population and wouldrequire screening of about 100 mutant clones to have a 98% chance ofbeing represented in the mutant population.

To identify the optimum amino acid at a position, mutants were screenedfor retention of enzymatic activity at high temperature. 100 tLI ofmedia was dispensed in each of the 96 wells of a microliter dish. Eachwell was inoculated with a Bacillus transformant and incubated at 37′with shaking. After 18 hours of growth, 20 μl of culture was dilutedinto 80 μl of 100 mM Tris-HCI, pH 8.0 in a second microliter dish. Thisdish was then incubated for one hour at 65° C. The dish was allowed tocool to room temperature following the high temperature incubation and100 μl of 1 mM SAAPF-pNA was added to each well. The wells which cleavedthe pNA (turned yellow) quickest were deemed to contain the most heatresistant subtilisin mutant. Once preliminary identification of a stablemutant was made from the second microliter dish, the Bacillus clone inthe corresponding well in the first microliter dish was grown up forfurther analysis.

The screening procedure identified stabilizing mutations at seven of theten positions which were examined. As noted, these amino acid positionswere selected at positions of the protein whose envirorunent has changedsubstantially by virtue of the calcium domain deletion. No mutationswere identified at positions 4, 74 and 214 which by themselvessignificantly increased the half-life of the mutant relative to theparent subtilisin. However, at position 214 the effect of onlyhydrophobic amino acids was screened. No mutations were found atpositions 5, 41 and 43 which resulted in measurable but modest increasesin stability. Moreover, several mutations were found at positions at 2,3, 73, and 206 which significantly increased the half-life of the mutantrelative to the parent subtilisin. These stabilizing mutations are shownin Table III as follows:

TABLE III Stabilizing Mutations Region of protein Site IncreaseN-terminus: Q2K 2.0-fold 63-85 α-helix: A73L 2.6-fold 202-220 β-ribbonQ206V 4.5-fold N-tenninus-β-ribbon S3C-Q206C (disulfide)  14-fold

Stabilizing amino acid modifications at positions 2(K), 73(L) and 206(V)were then combined to create subtilisin S73. The properties of S73subtilisin as well as S46, S79 and S86 are summarized in Table IV.

TABLE IV Specific Half-life Mutant Mutations¹ activity² (60 C)³ IncreaseS46 — 100 U/mg 2.3 min S73 Q2K 160 U/mg 25 min 11-fold A73L Q206V S79Q2K N.D.⁴ 18 min  8-fold A73L Q206C S86 Q2K, 85 U/mg 80 min 35-fold S3C⁵A73L Q206C⁵ ¹All of subtilisins S46, S73, S79 and S86 contain themutations M50F, Y217K and N218S and Q271E. ²Specific activity ismeasured againstsuccinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(SAAPF-pNA) in 10 mMTris-HCl, pH 8.0 at 25′C. ³Half-life is measured at 60′C in 10 mMTris-HCl, pH 8.0 50 mM NaCl and 10 mM EDTA. ⁴Not determined. ⁵Disulfidebond was formed between the cysteines at positions 3 and 206. Thefomiation of a disulfide bond was confirmed by measuring the radius ofgyration of the denatured protein by gel electrophoresis.

In many cases, the choice of amino acid at a particular position will beinfluenced by the amino acids at neighboring positions. Therefore, inorder to find the best combinations of stabilizing amino acids, it willbe necessary in some cases to vary the amino acids at two ormore-positions simultaneously. In particular, this was effected atpositions 3 and 206 with amino acids whose side chains can potentiallyinteract. It was determined that the best combination of modificationswas cysteine modifications at positions 3 and 206. This modification wasdenoted as S86. Because of the close proximity and suitable geometrybetween the cysteines at these two positions a disulfide cross-linkforms spontaneously between these two residues.

The stability of the S86 subtilisin was studied in relation to S73. Itwas found that the half-life of S86 is 80 minutes at 60° C. in 10 mMTris-HCl, pH 8-0, 50 mM NaCl and 10 mM EDTA, a 3.2-fold enhancementrelative to S73 subtilisin. By contrast, a 3-206 disulfide cross-linkwould not be able to form in native subtilisins which contain thecalcium A-site because the 75-83 binding loop separates the N-terminalamino acids from the 202-219 B ribbon. Therefore, the enhancement instability which occurs in the subject S86 mutant lacking the 75-83binding loop will likely not be observed with native subtilisinssimilarly cysteine modified at these positions.

It is expected that similar enhancement in stability will be inherent toother subtilisins of the I-S1 and I-S2 group if their calcium loops weredeleted (see Siezen et al, Protein Enizineering, 4, pp. 719-737 at FIG.7). This is a reasonable expectation based on the fact that the primarycalcium site in these different. subtilisins are formed from almostidentical 9 residue loops comprised in the identical position of helixC.

X-ray structures of the I-S1 subtilisins BPN and Carlsberg, as well asthe I-S2 subtilisin (savinase), have been determined to high resolution.Comparison of these structures demonstrates that all three have almostidentical calcium A-sites.

The x-ray structure of the class I subtilase, thermitase fromThermoactinomyces vulgaris, is also known. Though the overall homologyof BPN′ to thermitase is much lower than the homology of BPN′ to I-S1and I-S2 subtilisins, therrnitase has been shown to have an analogouscalcium A-site. In the case of thermitase, the loop is a ten residueinterruption at the identical site in helix C.

Thus, it is expected that the stabilizing mutations exemplified hereinwill impart similar beneficial effects on stability for the calciumloop-deleted versions of other class I subtilases.

The stability of S73, S76 and S86 subtilisins relative to S46 subtilisinwas compared by measuring their resistance to thermal inactivation at60° C. in 10 mM Tris-HCI, pH 8.0, 50 mM NaCl and 10 mM EDTA. Aliquotswere removed at intervals and the activity remaining in each aliquot wasdetermined. Under these conditions, the half-life of S46 subtilisin is2.3 minutes and the half-life of S73 is 25 minutes (Table IV).

In order to identify other mutants having increased stability anymutagenesis technique known by those skilled in the art may be used. Oneexample of such a technique for generating and screening subtilisinvariants involves three steps: 1) in vitro mutagenesis of the clonedsubtilisin gene; 2) expression of mutated genes in B. subtilis, and 3)screening for enhanced stability. The key element in the randommutagenesis approach is being able to screen large numbers of variants.

Although random mutagenesis may be employed, the mutagenesis proceduredescribed above allows for mutations to be directed to localized regionsof the protein (e.g., the N-terminal region). As noted supra, the S46,S47, S68, S73, S79 and S86 mutants (which comprise the active-site S221)were found to be enzymatically active. It is expected that othersubstitutions may be identified which provide for equivalent or evengreater stability and activity.

The activities of examples of the calcium-free subtilisin mutants of thepresent invention against the substrate sAAPF-pNA in Tris-HCl, pH 8.0and 25° C. are given in Table V as follows:

TABLE V Specific Half-life Subtilisin activity (55° C.) BPN′ 80 U/mg 2min S12 0.0025 U/mg N.D.¹ S15 0.0025 U/mg N.D.¹ S39 0.0025 U/mg N.D.¹S46 125 U/mg 22 min S47 90 U/mg 4.7 min S68 ˜100 U/mg 25 min ¹Half-liveswere not determined for inactive subtilisins.

As shown above, the subtilisin mutants S46, S47, S68, S73, S79 and S86have enhanced catalytic activity in comparison with subtilisin BPN′.Changes in catalytic efficiency due to the deletion were not expectedbecause of the fact that the active site of subtilisin is spatiallydistant from the calcium A-site.

The stability of these mutant subtilisins was compared to nativesubtilisin BPN′ by measuring their resistance to thermal inactivation.Since the stability of the calcium-free subtilisin mutants should beunaffected by metal chelating agents, the experiment was carried out inEDTA. Thermal inactivation in EDTA is a two step process as shown in thefollowing mechanism:

N(Ca)+EDTA⇄N+Ca:EDTA→U→I

The rate of calcium dissociation with the rate of unfolding as afunction of temperature for an inactive variant of subtilisin BPN′ wascompared in Bryan et al (Biochem. 31:49374945 (1992)). Repartitioning ofcalcium from site A into a strong chelator occurs at a rate 5 hour ⁻¹ at45° C. The kinetic barrier to calcium removal is 23 kcal/mol. Calcium isa integral part of the subtilisin structure and its association ordissociation requires significant but transient disruption insurrounding protein-protein interactions. This disruption in structurewould explain the high activation energy and slow kinetics of calciumbinding and dissociation. For example, breaking main-chain hydrogenbonds between the N-terminal region and the 75-83 loop region wouldallow the relatively buried calcium a passageway into or out of theprotein. Global unfolding in 10 M EDTA at 45° C. is much slower thancalcium dissociation, however, occurring at a rate of 0.04 hour ⁻¹, withan activation energy of ˜60 kcal/mol. Thus the predominant mechanism ofinactivation in EDTA is calcium dissociation followed by unfolding andloss of activity.

Because calcium binding reaches equilibrium quickly relative to the rateof unfolding, mutations which stabilize in EDTA must stabilizeapo-subtilisin. Increasing the binding constant for one of the calciumsites would not help unless the increase in binding affinity wereenormous. Consider a typical experiment in which 1 mM EDTA is added to100 μg/ml subtilisin (3.6 μM) bound to a stoichiometric amount ofcalcium. The calcium will partition between subtilisin and EDTAaccording to the equation:

[SCa]/[S_(total)]=K_(S-Ca)[S]/(1+K_(S-Ca)[S]+K_(E-Ca)[E])

where [SCa]/[S_(total)] is the fraction of subtilisin bound to calcium,[S]˜total subtilisin and [E]˜total EDTA. Since the binding constant ofsubtilisin for calcium at site A (K_(S-Ca))=7×10⁶ M⁻¹ and the bindingconstant of EDTA for calcium (K_(E-Ca))=2×10⁸ M⁻¹, then less than 0.02%subtilisin would be bound to calcium at equilibrium. Examples ofmutations which stabilize apo-subtilisin are M50F and the disulfidesC22-C87 and C206-C216 (Pantoliano et al, Biochem. 28:7205-7213 (1989)).The irony is that a mutation which preferentially stabilizesapo-subtilisin relative to the bound form, will weaken calcium bindingand catalyze inactivation under conditions of excess calcium and hightemperature (see the mechanism below). This phenomenon is displayed inthe M50F mutant, which is more stable than wild type in 10 mM EDTA butless stable in 10 mM CaCl₂.

The experiment to determine stability of the calcium-free subtilisinmutants was carried out in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 10 mMEDTA (the association constant of EDTA for calcium is 2×10⁸ M⁻¹). Theproteins were dissolved in this buffer and heated to 55° C. Aliquotswere removed at intervals and the activity remaining in each aliquot wasdetermined. The kinetics of inactivation are plotted in FIG. 10. Underthese conditions, the half-life of the subtilisin mutants was muchimproved over that of subtilisin BPN′. These results indicate thatsubtilisins which have been mutated to eliminate calcium binding at siteA have full catalytic activity and improved stability in EDTA relativeto subtilisin BPN′. A reasonable level of stability in S46 was achievedeven without additional mutations to compensate for lost interactionsresulting from deleting amino acids 75-83.

The inactivation of subtilisin in excess calcium is diagramed in thefollowing mechanism:

Ka (site B) Ka (site A) N(2Ca) N(Ca) + Ca N + 2Ca ↓k₁ ↓k₂ ↓k₃ U U U ↓ >>k₃ I

In excess calcium (e.g. ≧1 mM) and moderate temperature, calcium bindingand dissociation is in rapid equilibrium because calcium binding is muchfaster than unfolding. The rate of inactivation is determined by thefraction of each native species times its unfolding rate. Using theabove mechanism, one can show that calcium dependent stabilization ofsubtilisin is dominated by site A rather than site B. The rate ofinactivation of BPN′ at 65° C. as a function of calcium concentrationfits the data to the following mechanism:

33M⁻¹ 2.5 × 10⁵M⁻¹ N(Ca₂) N(Ca) + Ca N + 2Ca ↓0.0035 s⁻¹ ↓0.0085 s⁻¹↓8.7 s⁻¹ U U U ↓ > 25 s⁻¹ I

The mechanism predicts that K_(a)'s of site A and site B are 2.5×10⁵ M⁻¹and 33 M⁻¹ at 65°. The rate of inactivation of subtilisin with only siteA occupied (NCa) is about 1000-times slower than apo-subtilisin (N) andthe rate of inactivation with both sites occupied (NCa₂) is about2.5-times slower than with only site A occupied.

The second prediction has been borne out by measuring the calciumdependent stability of a mutant which has site B but lacks site A(Strausberg et al, Bio/Tech. 13:669-673 (1995)). The calcium-bindingloop is formed from a nine amino acid bubble in the last turn of a14-residue α-helix involving amino acids 63-85 (McPhalen & James,Biochem. 27:6582-6598 (1988)). Deleting amino acids 75-83 creates anuninterrupted helix and abolishes the calcium binding potential at siteA (Almog et al, Proteins 31:21-32 (1998); Bryan et al, Biochem.31:49374945 (1992)). The x-ray structure has shown that except for theregion of the deleted calcium-binding loop, the structure of the mutantand wild type protein are remarkably similar considering the size of thedeletion. The structures of subtilisin with and without the deletionsuperimpose with an rms difference between 261 Cα positions of 0.17 Å.The N-terminus of the wild-type protein lies beside the site A loop,furnishing one calcium coordination ligand, the side chain oxygen of Q2.In Δ75-83 subtilisin, the loop is gone, but the helix is uninterruptedand shows normal helical geometry over its entire length. The rate ofinactivation of Δ75-83 subtilisin is only 2.4-times slower in 10 mMCaCl₂, 50 mM NaCl than in 10 mM EDTA, 50 mM NaCl.

Another prediction of this last mechanism is that any mutations whichstabilize only in the presence of calcium will increase the bindingconstant for calcium to one or both of the calcium sites. This can beeither through effects on the binding sites themselves, as proposed formutations A116E, G131D, P172D, S63D, N76D and S78D (Pantoliano et al,Biochem. 28:7205-7213 (1989); Pantoliano et al, Biochem. 27:8311-8317(1988); Rollence et al, CRC Crit. Rev. Biotech. 8:217-224 (1988)), orthrough indirect effects on conformational stability as seen formutations S9A, I31L, S53T, L126I, E 156S, G166S, G169A, S188P and T254A.The indirect effect on calcium binding arises because apo-subtilisindisplays a loss of cooperativity in the unfolding reaction (Bryan et al,Biochem. 31:49374945 (1992)). Thus many mutations which stabilize in thepresence of calcium do not stabilize in the presence of EDTA, becausethey do not influence the rate determining step in the unfolding ofapo-subtilisin. In fact, most mutations in natural subtilisinsidentified to date stabilize only in the presence of calcium. Thesemutants increase calcium binding affinity because they preferentiallystabilize NCa relative to N. The premise that the effects of this classof mutations indirectly increase calcium affinity by increasing generalstability was tested using S88, a stabilize version of 75-83 subtilisin(Strausberg et al. 1995). The mutations S9A, I31L, E156S, G166S, G169A,N212G, S188P, K217L and T254A were introduced into the S88 version ofΔ75-83 subtilisin (see Table VI).

TABLE VI Increase in stabilization over S88 Mutation subtilisin ineither calcium or EDTA S9A 1.8 I31L 1.5 L126I 2.0 E156S 1.2 G166S 2.3G169A 5.0 N212G 1.5 M222Q 2.0 S188P 1.3 T254A 3.3 Combined in S88 1000subtilisin

Because the unfolding of the S88 subtilisin is cooperative in EDTA,these mutations now stabilize subtilisin S88, independent of calciumconcntration, to approximately the same extent that they stabilizesubtilisin BPN′ in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM CaCl₂.

Thus, the present inventors have provided convincing evidence thatsubtilisin mutants may be obtained which remain active and yet do notbind calcium. It is expected therefore that these mutants may beutilized in industrial environments that comprise chelating agents.While this has only been specifically shown with subtilisin BPN′,equivalent mutations should work with other serine proteases as well,most particularly other I-S1 or I-S2 subtilisins given that thesesubtilisins possess substantial sequence similarity, especially in thecalcium binding site.

Such strategies, for example, may involve comparing the sequence ofsubtilisin BPN′ to other serine proteases in order to identify the aminoacids which are suspected to be necessary for calcium binding and thenmaking suitable modifications, e.g., by site-specific mutagenesis. Sincemany subtilisins are related to subtilisin BPN′ not only through theirprimary sequences and enzymological properties, but also by X-raycrystallographic data, it is expected that other active subtilisinmutants which lack calcium binding may be produced by site specificmutagenesis. For example, structural evidence exists that the homologousenzyme subtilisin Carlsberg also comprises two calcium binding sites.Similarly, the X-ray structure of thermitase is known and thissubtilisin has an analogous calcium A binding-site to that of subtilisinBPN′. For thermitase, the calcium binding loop is a ten residueinterruption at the identical site in helix C. Accordingly, theseenzymes should also be amenable to the mutations described herein whicheliminate the calcium binding site and produce a stable, active enzyme.Moreover, as discussed supra, Siezen et al, has demonstrated that theprimary calcium binding site in all subtilisins in groups I-S1 and I-S2are formed from almost identical nine residue loops in the identicalposition of helix C. Thus, in view of the almost identical structures ofthe calcium A-sites, the methods described herein should be applicableto most if not all of the subtilisins in groups I-S1 and I-S2 set forthin Siezen et al.

Alternatively, if the amino acids which comprise the calcium bindingsites are already known for a particular subtilisin, the correspondingDNA will be mutated by site specific mutagenesis to delete one or moreof such amino acids, or to provide substitution, deletion or additionmutations which eliminate calcium binding.

The subject mutant subtilisins will generally be produced by recombinantmethods, in particular by expression of a subtilisin DNA which has beenmutated such that upon expression it results in a subtilisin proteinwhich is enzymatically active and which does not bind calcium.

Preferably, the subtilisin DNA's will be expressed in microbial hostcells, in particular Bacillus subtilis, because this bacteria naturallyproduces subtilisin, is an efficient secretor of proteins, and is ableto produce the protein in an active conformation. However, the inventionis not restricted to the expression of the subtilisin mutant inBacillus, but rather embraces expression in any host cell which providesfor expression of the desired subtilisin mutants. Suitable host cellsfor expression are well known in the art and include, e.g., bacterialhost cells such as Escherichia coli, Bacillus, Salmonella, Pseudomonas;yeast cells such as Saccharomyces cerevisiae, Pichia pastoris,Kluveromyces, Candida, Schizosaccharomyces; and mammalian host cellssuch as CHO cells. Bacterial host cells, however, are the preferred hostcells for expression.

Expression of the subtilisin DNA will be provided using availablevectors and regulatory sequences. The actual selection will depend inlarge part upon the particular host cells which are utilized forexpression. For example, if the subtilisin mutant DNA is expressed inBacillus, a Bacillus promoter will generally be utilized as well as aBacillus derived vector. The present inventors in particular used thepUB110-based expression vector and the native promoter from thesubtilisin BPN′ gene to control expression on Bacillus subtilis.

It is further noted that once the amino acid sequence of a particularsubtilisin mutant which does not bind calcium has been elucidated, itmay also be possible to make the subtilisin mutant by protein synthesis,e.g., by Merrifield synthesis. However, expression of the subtilisinmutants in microbial host cells will generally be preferred since thiswill allow for the microbial host cell to produce the subtilisin proteinin a proper conformation for enzymatic activity. However, since thepresent inventors further teach herein a method for obtaining in vitrorefolding of the subtilisin mutant, it should be possible to convertimproperly folded subtilisin mutants into an active conformation.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat the same is intended only as illustrative and in nowise limitative.

EXAMPLES Example 1

Cloning and Expression The subtilisin gene from Bacillusamyloliquefaciens (subtilisin BPN′) has been cloned, sequenced, andexpressed at high levels from its natural promoter sequences in Bacillussubtilis (Wells et al., Nucleic Acids Res. 11:7911-7925 (1983); Vasanthaet al., J. Bacteriol. 159:811819(1984)). All mutant genes were reclonedinto a pUB110-based expression plasmid and used to transform B.subtilis. The B. subtilis strain used as the host contains a chromosomaldeletion of its subtilisin gene and therefore produces no backgroundwild type (wt) activity (Fahnestock et al., Appl. Environ. Microbial.53:379-384 (1987)). Oligonucleotide mutagenesis was carried out aspreviously described. (Zoller et al., Methods Enzymol. 100:468-500(1983); Bryan et al., Proc. Natl. Acad. Sci. 83:3743-3745 (1986b)).S221C was expressed in a 1.51 New Brunswick fermentor at a level ofapproximately 100 mg of the correctly processed mature form per liter.The addition of wild type subtilisin to promote production of the matureform of S221C subtilisin was not required in our bacillus host strain aswas the case for prior strains (Abrahmsen et al., Biochemistry30:4151-4159 (1991)).

Protein Purification & Characterization. Wild type subtilisin and thevariant enzymes were purified and verified for homogeneity essentiallyas described in Bryan et al., Proc. Natl. Acad. Sci. 83:3743-3745(1986b); Pantoliano et al., Biochemistry 26:2077-2082 (1987); andBiochemistry 27:8311-8317 (1988). In some cases the C221 mutantsubtilisins were re-purified on a sulfhydryl specific mercury affinitycolumn (Affi-gel 501, Biorad). Assays of peptidase activity wereperformed by monitoring the hydrolysis ofsuccinyl-(L)Ala-(L)-Ala-(L)-Pro-(L)-Phe-p-nitroanilide, hereinaftersAAPFna, as described by DelMar et al., Anal Biochem. 99:316-320 (1979).The protein concentration, [P], was determined using P^(0.1%)=1.17 at280 mn (Pantoliano et al, Biochemistry 28:7205-7213 (1989)). Forvariants which contain the Y217K change, the p^(0.1%) at 280 nm wascalculated to be 1.15 (or 0.96×X wt), based on the loss of one Tyrresidue (Pantoliano et al., Biochemistry 28:7205-7213 (1989)).

N-terminal Analysis The first five amino acids of subtilisin S 15 weredetermined by sequential Edman degradation and HPLC analysis. Thisrevealed that 100% of the material had the amino acid sequence expectedfrom the DNA sequence of the gene and that processing of the pro-peptidewas at the same position in the sequence for the mutant as for the wildtype enzyme.

Example 2

Structure of the calcium A site of S12 subtilisin Calcium at site A iscoordinated by five carbonyl oxygen ligands and one aspartic acid. Fourof the carbonyl oxygen ligands to the calcium are provided by a loopcomposed of amino acids 75-83 (FIG. 2). The geometry of the ligands isthat of a pentagonal bipyramid whose axis runs through the carbonyls of75 and 79. On one side of the loop is the bidentate carboxylate (D41),while on the other side is the N-terminus of the protein and the sidechain of Q2. The seven coordination distances range from 2.3 to 2.6 A,the shortest being to the aspartyl carboxylate. Three hydrogen bondslink the N-terminal segment to loop residues 78-82 in parallel-betaarrangement.

Preparation of apo-subtilisin S11 and S12 subtilisin contain an equalmolar amount of tightly bound calcium after purification. X-raycrystallography has shown this calcium to be bound to the A site (Finzelet al., J. Cell. Biochem. Suppl. 10A:272 (1986); Pantoliano et al.,Biochemistry 27:8311-8317 (1988); McPhalen et al., Biochemistry27:6582-6598 (1988)).

Complete removal of calcium from subtilisin is very slow, requiring 24hours of dialysis against EDTA at 25° C. to remove all calcium from theprotein and then 48 more hours of dialysis in high salt (Brown et al.,Biochemistry 16:3883-3896 (1977)) at 4° C. to remove all EDTA from theprotein. To prepare the calcium-free form of subtilisins S 11 and S12,20 mg of lyophilized protein was dissolved in 5 ml of 10 mM EDTA, 10 mMtris(hydroxymethyl)amino-methane hydrochloric acid (hereinafterTris-HCl) at pH 7.5 and dialyzed against the same buffer for 24 hours at25° C. In order to remove EDTA, which binds to subtilisin at low ionicstrength, the protein was then dialyzed twice against 2 liters of 0.9MNaCl, 10 mM Tris-HCl at pH 7.5 at 4° C. for a total of 24 hours and thenthree times against 2 liters of 2.5 mM Tris-HCl at pH 7.5 at 4° C. for atotal of 24 hours. Chelex 100 was added to all buffers not containingEDTA. When versions of C221 subtilisin not containing stabilizing aminoacid substitutions were used, up to 50% of the protein-precipitatedduring this procedure. It is essential to use pure native apoenzyme intitration experiments so that spurious heat produced by precipitationupon the addition of calcium does not interfere with the measurement ofthe heat of binding.

To ensure that preparations of apo-subtilisin were not contaminated withcalcium or EDTA, samples were checked by titration with calcium in thepresence of Quin2 prior to performing titration calorimetry.

Titration Calorimetry Measurements The calorimetric titrations wereperformed with a Microcal Omega titration calorimeter as described indetail by Wiseman et al., Analytical Biochemistry 179:131-137 (1989).The titration calorimeter consists of a matched reference cellcontaining the buffer and a solution cell (1.374 mL) containing theprotein solution. Microliter aliquots of the ligand solution are addedto the solution cell through a rotating stirrer syringe operated with aplunger driven by a stepping motor. After a stable baseline was achievedat a given temperature, the automated injections were initiated and theaccompanying heat change per injection was determined by a thermocouplesensor between the cells. During each injection, a sharp exothermic peakappeared which returned to the baseline prior to the next injectionoccurring 4 minutes later. The area of each peak represents the amountof heat accompanying binding of the added ligand to the protein. Thetotal heat (Q) was then fit by a nonlinear least squares minimizationmethod (Wiseman et al., Analytical Biochemistry 179:131-137 (1989)) tothe total ligand concentration, [Cal_(total), according to the equation:

dQ/d[Ca]_(total)=ΔH[1/2+(1−(1+r)/2−Xr/2)/Xr−2Xr(1−r)+1+r²)^(1/2)]  (1)

wherein 1/r=[P]_(total)xK_(a) and X_(r)=[Ca]_(total)/[P]_(total).

The binding of calcium to the S11 and S12 subtilisins was measured bytitration calorimetry as it allows both the binding constant and theenthalpy of binding to be determined (Wiseman et al., AnalyticalBiochemistry 179:131-137 (1989); Schwarz et al., J. Biol. Chem.266:24344-24350 (1991)).

The S 11 and S12 subtilisin mutants were used in titration experimentsbecause production of the wild type apoenzyme is impossible due to itsproteolytic activity and low stability. Titrations of S11 and S12 wereperformed at protein concentrations [P]=30 μM and 100 μM. Titration ofthe S11 apoenzyme with calcium at 25° C. is shown in FIG. 4. The datapoints correspond to the negative heat of calcium binding associatedwith each titration of calcium. The titration calorimeter is sensitiveto changes in Ka under conditions at which the product of K_(a)×[P] isbetween 1 and 1000 (Wiseman et al., Analytical Biochemistry 179:131-137(1989)). Since the K_(a) for subtilisin is about 1×10⁷ M⁻¹, theseprotein concentrations result in values of K_(a)×[P]=300 and 1000. Atlower protein concentrations the amount of heat produced per titrationis difficult to measure accurately.

The results of fitting the titrations of S11 and S12 to a calculatedcurve are summarized in Table 2. The parameters in the table includebinding parameters for stoichiometric ratio (n), binding constant(K_(a)) and binding enthalpy (ΔH). These parameters were determined fromdeconvolution using nonlinear least squares minimization (Wiseman etal., Analytical Biochemistry 179:131-137 (1989)). Measurements for eachexperimental condition were performed in duplicate at 25° C. The proteinconcentrations ranged from 30 to 100 μM while the concentration of thecalcium solutions were about 20 times the protein concentrations. Eachbinding constant and enthalpy were based on several titration runs atdifferent concentrations. Titration runs were performed until thetitration peaks were close to the baseline.

TABLE 2 Titration Calorimetry of the Calcium A Site in SubtilisinMutants S11 and S12. Parameters calculated from fit Mutant [P] n K_(a)ΔH S11 100 μM 0.98 ± 0.01 7.8 ± 0.2 × 10⁶ −11.3 ± 0.1 S11  33 μM 0.9 ±0.3 6.8 ± 1.5 × 10⁶ −10.9 ± 0.2 S12 100 μM 0.99 ± 0.01 6.4 ± 0.2 × 10⁶−11.8 ± 0.5

The average values obtained are similar for S11 and S12: ΔH=˜−11kcal/mol; K_(a)=7×10⁶ M⁻¹ and a stoichiometry of binding of 1 calciumsite per molecule. The weak binding site B does not bind calcium atconcentrations below the millimolar range, and therefore does notinterfere with measurement of binding to the binding site A. Thestandard free energy of binding at 25° C. is 9.3 kcal/mol. The bindingof calcium is therefore primarily enthalpically driven with only a smallnet loss in entropy (ΔS_(binding)=−6.7 cal/° mol).

Example 3

In vitro refolding of S15 subtilisin. For refolding studies subtilisinwas maintained as a stock solution in 2.5 mM Tris-HCl at pH 7.5 and 50mM KCl at a concentration of approximately 100 μM. The protein wasdenatured by diluting the stock solution into 5M guanidine hydrochloride(Gu-HCl) at pH 7.5 or in most cases into 25 mM H₃PO₄ or HCl at pH1.8-2.0. The fmal concentration of protein was 0.1 to 5 μM. S15 wascompletely denatured in less than 30 seconds by these conditions. S12required approximately 60 minutes to become fully denatured.Acid-denatured protein was then neutralized to pH 7.5 by the addition ofTris-base (if denatured in HCl) or 5M NaOH (if denatured in H₃PO₄).Refolding was initiated by the addition of KCl, NaCl or CaCl₂ to thedesired concentration. For example, KCl was added from a stock solutionof 4M to a final concentration of 0.1 to 1.5M with rapid stirring. Inmost cases renaturation was carried out at 25° C. The rate ofremturation was determined spectrophotometrically by uv absorption fromthe increase in extinction at λ=286, from the increase in intrinsictyrosine and tryptophan fluorescence (excitation λ=282, emission λ−347),or by circular dichroism from the increase in negative ellipticity atλ=222 nm.

Example 4

X-ray Crystallography. Large single crystal growth and X-ray diffractiondata collection were performed essentially as previously reported (Bryanet al., Proteins: Struct. Funct. Genet. 1:326-334 (1986a); Pantoliano etal., Biochemistry 27:8311-8317 (1988); Pantoliano et al., Biochemistry28:7205-7213 (1989)) except that it was not necessary to inactivate theS221C variants with diisopropyl fluorophosphate (DFP) in order to obtainsuitable crystals. The starting model for S12 was made from thehyperstable subtilisin mutant 8350 (Protein Data Bank entry ISO1.pdb).The S12 structure was refmed and then modified to provide the startingmodel for S15.

Data sets with about 20,000 reflections between 8.0 Å and 1.8 Åresolution were used to refine both models using restrainedleast-squares techniques (Hendrickson et al., “Computing inCrystallography” in Diamond et al., eds., Bangalore: Indian Institute ofScience 13.01-13.23 (1980)). Initial difference maps for S15, phased bya version of S12 with the entire site A region omitted, clearly showedcontinuous density representing the uninterrupted helix, permitting aninitial S15 model to be constructed and refinement begun. Each mutantwas refmed from R approximately 0.30 to R approximately 0.18 in abouteighty cycles, interspersed with calculations of electron density mapsand manual adjustments using the graphics modeling program FRODO (Jones,J. Appl. Crystallogr. 11:268-272 (1978)).

Except for the region of the deleted calcium-binding loop, thestructures of S12 and S15 are very similar, with a root mean square(r.m.s) deviation of 0.18 Å between 262 α-carbons. The N-terminus of S12(as in the wild-type) lies beside the site A loop, furnishing onecalcium coordination ligand, the side chain oxygen of Q2. In S15 theloop is gone, leaving residues 1-4 disordered. In S12 (as in wild type)the site A loop occurs as an interruption in the last turn of a14-residue alpha helix; in S15 this helix is uninterrupted and showsnormal helical geometry over its entire length. Diffuse differencedensity and higher temperature factors indicate some disorder in thenewly exposed residues adjacent to the deletion.

Example 5

Differential Scanning Calorimetry The stability properties of S12 andS15 were studied using DSC (differential scanning calorimetry). TheΔ75-83 mutant (S15) is very similar in melting temperature to theapoenzyme of S12. The DSC profiles of apo-S12 and S15 are shown in FIG.3. The temperature of maximum heat capacity is 63.0° C. for S15 and63.5° C. for apo-S12 at pH 9.63. The DSC experiments were carried out athigh pH to avoid aggregation during the denaturation process. The amountof excess heat absorbed by a protein sample as the temperature increasedthrough a transition from the folded to unfolded state at constantpressure, which provided a direct measurement of the AH of unfolding(Privalov et al., Methods Enzymol. 131:4-51 (1986)). ΔH_(cal) ofunfolding for apo-S12 and S15 is about 140 kcal/mol. Above pH 10.0, theunfolding transition for S15 fit a two-state model reasonably well,consistent with equilibrium thermodynamics as expressed in the van'tHoff equation (dln K/dT=ΔH_(vH)/(RT²)) with ΔH_(vH) (the van't Hoffenthalpy or apparent enthalpy) approximately equal to ΔH_(cal) (thecalorimetric or true enthalpy). At pH 9.63, however, the melting profilefor both proteins was asymmetric indicating that the unfolding is not apure two-state process.

Example 6

Measuring kinetics of calcium dissociation. The dissociation of calciumfrom subtilisin is a slow process. To measure this rate the fluorescentcalcium chelator Quin 2 was used. Quin 2 binds calcium with a K_(a) of1.8×10⁸ at pH 7.5 (Linse et al., Biochemistry 26:6723-6735 (1987)). Thefluorescence of Quin 2 at 495 nm increases by approximately 6-fold whenbound to calcium (Bryant, Biochem. J. 226:613-616 (1985)). SubtilisinS11 or S12 as isolated contains one calcium ion per molecule. When mixedwith an excess of Quin 2, the kinetics of calcium release from theprotein can be followed from the increase in fluorescence at 495 nm. Thereaction is assumed to follow the pathway N(Ca)N+Ca+Quin 2Quin(Ca). Thedissociation of calcium front subtilisin is very slow relative tocalcium binding by Quin 2, such that the change in fluorescence of Quin2 is equal to the rate of calcium dissociation from subtilisin. As canbe seen in FIG. 5a, the initial release of calcium from S11 followssimple first order kinetics.

Temperature dependence of calcium dissociation The first order rateconstant (k) for calcium dissociation was measured from 200 to 45° C.The plot of ln k vs. 1/T°K is roughly linear. The calcium dissociationdata was curve fit using transition state theory according to the Eryingequation:

ΔG=−RT ln K=−RT ln kh/k_(B)T  (2)

wherein k_(B) is the Boltzman constant, h is Planck's constant and k isthe first order rate constant for folding. A graph of ln hk/k_(B)T vs.1/T is shown in FIG. 5b.

The data was then curve fit according to the equation (Chen et al.,Biochemistry 28:691-699 (1989)):

ln K=A+B(To/T)+C ln (To/T)  (3)

wherein A=[ΔCp+ΔS+(To)]/R; B=A−ΔG(T_(o))/RTo; C=ΔCp/R. The data obtainedyields the following results: ΔG=22.7 kcal/mol; ΔCp′=−0.2 kcal/°mol;ΔS′=−10 cal/°mol; and ΔH′=19.7 kcal/mol at a reference temperature of25° C. A possible slight curvature of the plot would be due to a changein heat capacity associated with formation of the transition state(ΔCp′=0.2 kcal/°mol). ΔCp for protein folding has been shown to beclosely correlated with a change in exposure of hydrophobic groups towater (Privalov et al., Adv. Protein Chem. 39:191-234 (1988);Livingstone et al., Biochemistry 30:4237-4244 (1991)). In terms of heatcapacity, the transition state therefore appears similar to the nativeprotein. The values for ΔS′ and ΔH′ obtained from FIG. 5b indicate thatthe transition state is enthalpically less favorable than the calciumbound form with only a small change in entropy.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

All references cited herein are incorporated in their entirety, as ifindividually incorporated by reference.

2 1 1868 DNA Bacillus amyloliquefaciens CDS (450)..(1595) 1 tttttccgcaattatatcat tgacaatatc aacatcaatg atattcatta tcattatttt 60 tataaaatggtttcacagct tttctcggtc aagaaagcca aagactgatt tcgcttacgt 120 ttccatcagtcttctgtatt caacaaaaga tgacatttat cctgtttttg gaacaacccc 180 caaaaatggaaacaaaccgt tcgacccagg aaacaagcga gtgattgctc ctgtgtacat 240 ttactcatgtccatccatcg gttttttcca ttaaaattta aatatttcga gttcctacga 300 aacgaaagagagatgatata cctaaataga aataaaacaa tctgaaaaaa attgggtcta 360 ctaaaatattattccatact atacaattaa tacacagaat aatctgtcta ttggttattc 420 tgcaaatgaaaaaaaggaga ggataaaga gtg aga ggc aaa aaa gta tgg atc 473 Val Arg Gly LysLys Val Trp Ile -105 -100 agt ttg ctg ttt gct tta gcg tta atc ttt acgatg gcg ttc ggc agc 521 Ser Leu Leu Phe Ala Leu Ala Leu Ile Phe Thr MetAla Phe Gly Ser -95 -90 -85 aca tcc tct gcc cag gcg gca ggg aaa tca aacggg gaa aag aaa tat 569 Thr Ser Ser Ala Gln Ala Ala Gly Lys Ser Asn GlyGlu Lys Lys Tyr -80 -75 -70 att gtc ggg ttt aaa cag aca atg agc acg atgagc gcc gct aag aag 617 Ile Val Gly Phe Lys Gln Thr Met Ser Thr Met SerAla Ala Lys Lys -65 -60 -55 aaa gat gtc att tct gaa aaa ggc ggg aaa gtgcaa aag caa ttc aaa 665 Lys Asp Val Ile Ser Glu Lys Gly Gly Lys Val GlnLys Gln Phe Lys -50 -45 -40 tat gta gac gca gct tca gct aca tta aac gaaaaa gct gta aaa gaa 713 Tyr Val Asp Ala Ala Ser Ala Thr Leu Asn Glu LysAla Val Lys Glu -35 -30 -25 -20 ttg aaa aaa gac ccg agc gtc gct tac gttgaa gaa gat cac gta gca 761 Leu Lys Lys Asp Pro Ser Val Ala Tyr Val GluGlu Asp His Val Ala -15 -10 -5 cat gcg tac gcg cag tcc gtg cct tac ggcgta tca caa att aaa gcc 809 His Ala Tyr Ala Gln Ser Val Pro Tyr Gly ValSer Gln Ile Lys Ala -1 1 5 10 cct gct ctg cac tct caa ggc tac act ggatca aat gtt aaa gta gcg 857 Pro Ala Leu His Ser Gln Gly Tyr Thr Gly SerAsn Val Lys Val Ala 15 20 25 gtt atc gac agc ggt atc gat tct tct cat cctgat tta aag gta gca 905 Val Ile Asp Ser Gly Ile Asp Ser Ser His Pro AspLeu Lys Val Ala 30 35 40 45 ggc gga gcc agc atg gtt cct tct gaa aca aatcct ttc caa gac aac 953 Gly Gly Ala Ser Met Val Pro Ser Glu Thr Asn ProPhe Gln Asp Asn 50 55 60 aac tct cac gga act cac gtt gcc ggc aca gtt gcggct ctt aat aac 1001 Asn Ser His Gly Thr His Val Ala Gly Thr Val Ala AlaLeu Asn Asn 65 70 75 tca atc ggt gta tta ggc gtt gcg cca agc gca tca ctttac gct gta 1049 Ser Ile Gly Val Leu Gly Val Ala Pro Ser Ala Ser Leu TyrAla Val 80 85 90 aaa gtt ctc ggt gct gac ggt tcc ggc caa tac agc tgg atcatt aac 1097 Lys Val Leu Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile IleAsn 95 100 105 gga atc gag tgg gcg atc gca aac aat atg gac gtt att aacatg agc 1145 Gly Ile Glu Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn MetSer 110 115 120 125 ctc ggc gga cct tct ggt tct gct gct tta aaa gcg gcagtt gat aaa 1193 Leu Gly Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala ValAsp Lys 130 135 140 gcc gtt gca tcc ggc gtc gta gtc gtt gcg gca gcc ggtaac gaa ggc 1241 Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala Gly AsnGlu Gly 145 150 155 act tcc ggc agc tca agc aca gtg ggc tac cct ggt aaatac cct tct 1289 Thr Ser Gly Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys TyrPro Ser 160 165 170 gtc att gca gta ggc gct gtt gac agc agc aac caa agagca tct ttc 1337 Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln Arg AlaSer Phe 175 180 185 tca agc gta gga cct gag ctt gat gtc atg gca cct ggcgta tct atc 1385 Ser Ser Val Gly Pro Glu Leu Asp Val Met Ala Pro Gly ValSer Ile 190 195 200 205 caa agc acg ctt cct gga aac aaa tac ggg gcg tacaac ggt acg tca 1433 Gln Ser Thr Leu Pro Gly Asn Lys Tyr Gly Ala Tyr AsnGly Thr Ser 210 215 220 atg gca tct ccg cac gtt gcc gga gcg gct gct ttgatt ctt tct aag 1481 Met Ala Ser Pro His Val Ala Gly Ala Ala Ala Leu IleLeu Ser Lys 225 230 235 cac ccg aac tgg aca aac act caa gtc cgc agc agttta gaa aac acc 1529 His Pro Asn Trp Thr Asn Thr Gln Val Arg Ser Ser LeuGlu Asn Thr 240 245 250 act aca aaa ctt ggt gat tct ttc tac tat gga aaaggg ctg atc aac 1577 Thr Thr Lys Leu Gly Asp Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn 255 260 265 gta cag gcg gca gct cag taaaacataa aaaaccggccttggccccgc 1625 Val Gln Ala Ala Ala Gln 270 275 cggtttttta ttatttttcttcctccgcat gttcaatccg ctccataatc gacggatggc 1685 tccctctgaa aattttaacgagaaacggcg ggttgacccg gctcagtccc gtaacggcca 1745 agtcctgaaa cgtctcaatcgccgcttccc ggtttccggt cagctcaatg ccgtaacggt 1805 cggcggcgtt ttcctgataccgggagacgg cattcgtaat cggatcagaa gcaaaactga 1865 gca 1868 2 382 PRTBacillus amyloliquefaciens 2 Val Arg Gly Lys Lys Val Trp Ile Ser Leu LeuPhe Ala Leu Ala Leu -105 -100 -95 Ile Phe Thr Met Ala Phe Gly Ser ThrSer Ser Ala Gln Ala Ala Gly -90 -85 -80 Lys Ser Asn Gly Glu Lys Lys TyrIle Val Gly Phe Lys Gln Thr Met -75 -70 -65 -60 Ser Thr Met Ser Ala AlaLys Lys Lys Asp Val Ile Ser Glu Lys Gly -55 -50 -45 Gly Lys Val Gln LysGln Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr -40 -35 -30 Leu Asn Glu LysAla Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala -25 -20 -15 Tyr Val GluGlu Asp His Val Ala His Ala Tyr Ala Gln Ser Val Pro -10 -5 -1 1 5 TyrGly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr 10 15 20 ThrGly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser 25 30 35 SerHis Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser 40 45 50 GluThr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala 55 60 65 GlyThr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala 70 75 80 85Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser 90 95100 Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn 105110 115 Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala120 125 130 Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val ValVal 135 140 145 Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser SerThr Val 150 155 160 165 Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala ValGly Ala Val Asp 170 175 180 Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser ValGly Pro Glu Leu Asp 185 190 195 Val Met Ala Pro Gly Val Ser Ile Gln SerThr Leu Pro Gly Asn Lys 200 205 210 Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Ser Pro His Val Ala Gly 215 220 225 Ala Ala Ala Leu Ile Leu Ser LysHis Pro Asn Trp Thr Asn Thr Gln 230 235 240 245 Val Arg Ser Ser Leu GluAsn Thr Thr Thr Lys Leu Gly Asp Ser Phe 250 255 260 Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala Ala Ala Gln 265 270 275

What is claimed is:
 1. A subtilisin protein which has been mutated toeliminate the ability of said subtilisin protein to bind calcium at thecalcium A binding site wherein the mutated subtilisin protein comprisesa deletion of amino acids 75-83 and a deletion of part or all of theN-terminal region amino acids 1-22, wherein said amino acid positionsare numbered according to correspondence with the amino acid positionsof the amino acid sequence of subtilisin BPN′ set forth in SEQ ID NO. 1.2. The subtilisin protein of claim 1, wherein amino acids 1-22 of theN-terminal region are deleted.
 3. The subtilisin protein of claim 1,wherein amino acids 1-17 of the N-terminal region are deleted.
 4. Thesubtilisin protein of claim 1, wherein the subtilisin is from a Bacillusstrain.
 5. The subtilisin protein of claim 4, wherein the subtilisinmutant is a subtilisin BPN′ mutant, a subtilisin Carlsberg mutant, asubtilisin DY mutant, a subtilisin amylosacchariticus mutant or asubtilisin mesenticopeptidase mutant or a subtilisin Savinase mutant. 6.The subtilisin protein of claim 5, wherein the subtilisin mutant is asubtilisin BPN′ mutant.
 7. The subtilisin protein of claim 1, whereinsaid protein also contains one or more substitutions selected from thegroup consisting of S221C, Y217K, P5A, M50F, N218S, P5S, Q2K, A73L,Q206V, Q206C, S3C, K43N, Q217E, S9A, I31L, E156S, G166S, G169A, S188P,N212G, K217L, T254A, L126I, and M222Q.
 8. The subtilisin protein ofclaim 1, wherein one or more substitutions are also made at amino acidpositions: 41-45; 70-74; 86-87; 181-184; 200-214; 226-237; and 267-275.9. A recombinant method which provides for the expression of asubtilisin protein which has been mutated to eliminate the ability ofsaid subtilisin protein to bind calcium at the calcium A binding site,wherein the mutated subtilisin protein comprises a deletion of aminoacids 75-83 and a deletion of part or all of the N-terminal region aminoacids 1-22, wherein said amino acid positions are numbered according tocorrespondence with the amino acid positions of the amino acid sequenceof subtilisin BPN′ set forth in SEQ ID NO. 1, said method comprising:(a) transforming a recombinant host cell with an expression vectorcomprising a DNA sequence encoding an enzymatically active subtilisinwhich does not bind calcium and which has a deletion of part or all ofthe N-terminal region amino acids 1-22; (b) culturing said host cellsunder conditions which provide for the expression of the enzymaticallyactive subtilisin mutant; and (c) recovering the expressed enzymaticallyactive subtilisin mutant from said microbial host.
 10. The recombinantmethod of claim 9, wherein the subtilisin mutant is a subtilisin BPN′mutant.
 11. The recombinant method of claim 10, wherein the subtilisinBPN′ DNA comprises one or more additional mutations which provide forenhanced thermal stability or which provide for restoration ofcooperativity of folding of the subtilisin protein.
 12. The recombinantmethod of claim 9, wherein the mutated subtilisin protein also has oneor more substitutions selected from the group consisting of S221C,Y217K, P5A, M50F, N218S, P5S, Q2K, A73L, Q206V, Q206C, S3C, K43N, Q217E,S9A, I31L, E156S, G166S, G169A, S188P, N212G, K217L, T254A, L126I, andM222Q.
 13. The recombinant method of claim 9, wherein mutated subtilisinprotein also has a substitution at one of the following amino acidpositions: 41-45; 70-74; 86-87; 181-184; 200-214; 226-237, and 267-275.14. A recombinant DNA which encodes the mutated subtilisin protein ofclaim 1.